1. Field of the Invention
The present invention relates to a stable liquid formulation of a fusion protein having an Fc domain of a human immunoglobulin G (IgG). More particularly, the present invention relates to a liquid formulation having a stabilized protein in which an Fc domain of a human IgG and a soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor are fused, for example, aflibercept, to a composition for stabilizing a protein in which an Fc domain of an IgG and a soluble extracellular domain of a VEGF receptor are fused, and to a method for stabilizing a protein in which an Fc domain of an IgG and a soluble extracellular domain of a VEGF receptor are fused.
2. Discussion of Related Art
A type of cell-inducing dimer mitogen having selectivity to vascular endothelial cells has been identified, and is designated as vascular endothelial growth factor (VEGF). The VEGF is an important factor which increases angiogenesis and vascular permeability.
It has been known that the VEGF activates VEGF receptors (i.e., VEGFR-1, VEGFR-2, and VEGFR-3) which are membrane-spanning tyrosine kinase receptors. Among the VEGF receptors, VEGFR-1 and VEGFR-2 have 7 Ig-like sequences, a single transmembrane region, and a consensus tyrosine kinase region in the extracellular domain in order to bind to the VEGF. These features are applied as a sequence for an anti-VEGF agent. Aflibercept, which is an ophthalmic therapeutic agent, is a soluble decoy receptor of about 115 kDa (including glycosylation) having a structure in which a second binding domain of VEGFR-1 and a third binding domain of VEGFR-2 are fused with an Fc region of a human IgG1 (see [Drug Design Development and Therapy (2013), 3(7), 711-722]).
In mechanisms via the VEGF, abnormal angiogenesis is associated with ophthalmic diseases such as wet age-related macular degeneration, diabetic macular edema, and macular edema in retinal vein occlusion, etc. (see [J. Korean Med. Assoc. (2014), 57(7), 614-623]).
Examples of therapeutic agents for these ophthalmic diseases include pegaptanib (trade name: Macugen), ranibizumab (trade name: Lucentis), bevacizumab (trade name: Avastin), and aflibercept (trade name: Eylea). Aflibercept is approved for the treatment of wet age-related macular degeneration in 2011 (see [Biol. Ther. (2012), 2(3) 1-22], and [Drug Design Development and Therapy (2013), 3(7), 711-722], etc.). Among the therapeutic agents above, aflibercept has been reported to have the best therapeutic effect on patients with diabetic macular edema having advanced amblyopia (see [NEJM (2015), 372(13) 1193-1203]. Aflibercept has been commercialized as a therapeutic agent for metastatic colorectal cancers (trade name: Zaltrap) and a therapeutic agent for retinal vein occlusion, diabetic macular edema, choroidal neovascularization, and wet age-related macular degeneration (trade name: Eylea).
Physicochemical modifications occur in protein drugs including antibody drugs under the non-optimal condition. In particular, factors such as temperature, pH, concentration of a salt, contact with air, concentration of a protein, and types of buffers significantly affect oxidation, deamidation, isomerization, and polymerization, of a protein. These modifications cause aggregation, and generate fragments, isomers of the protein, so that bioactivity may be reduced. These properties differ among proteins. Particularly, for an Fc fusion protein, due to the problem in folding, separate 3 peaks appear in hydrophobic interaction chromatography (see [Antibodies (2013), 2,452-500]).
International Publication WO 2007/149334 discloses “an ophthalmic formulation including 1-100 mg/ml of aflibercept, 0.01-5% of an organic cosolvent (e.g., polysorbate, polyethylene glycol, propylene glycol, etc.), 30-150 mM of an isotonic agent (e.g., NaCl, KCl etc.), 5-40 mM of sodium phosphate buffer and 1.0-7.5% of stabilizer (e.g., sucrose, sorbitol, glycerol, trehalose, and mannitol, etc.)” and “a lyophilizable formulation including 5-50 mg/ml of aflibercept, 5-25 mM of sodium phosphate buffer, 0.01-0.15% of an organic cosolvent, 1-10% of a stabilizer, and 20-150 mM of an isotonic agent.” The formulation disclosed in International Publication WO 2007/149334 may be applied to a prefilled syringe suitable for intravitreal administration.
For the ophthalmic formulation and lyophilizable formulation disclosed in International Publication WO 2007/149334, an effect of inhibiting production of impurities and byproducts due to aggregation, fragmentation and isomerization of aflibercept, was reported. However, the formulation in International Publication WO 2007/149334 was problematic in that the effect of stabilizing aflibercept was markedly reduced under harsh conditions such as high temperature condition of 40° C. or more, or shaking condition.
Therefore, the present inventors have completed the present invention by developing a liquid formulation having enhanced stability under the harsh conditions as well as stably maintaining a fusion protein having an IgG Fc domain such as aflibercept under the storing condition for a long period of time.